Zeranol (Z) is a non-steroidal agent with estrogenic activity (E2) that is used as an anobolic growth promoter in the U.S. beef industries. Consumption of bio-active Z containing-beef has a potential risk in human normal and cancerous breast cells. Previous work suggests that PTP gamma (PTPG) is an E2-regulated breast cancer suppressor gene. Z is capable in down-regulating the PTPG both in mRNA and protein levels in human normal and cancerous breast tissues and cells. The overall HYPOTHESIS to be evaluated is that the consumption of beef products, derived from Z-implanted beef cattle may possess a potential health impact on human consumers, particularly with respect to reproductive endocrinology and hormone-sensitive breast tissues. Specific Aims: (1): Define epigenetic and gene expression alterations in primary cultured normal human breast epithelial cells, stromal cells, and tissues exposed to serum and meat extracts collected from Zimplanted beef heifers. (2): Define DNA methylation changes in the estrogen inducible mammary tumor ACI rat model exposed to bio-active Z-containing diets formulated from Z-implanted beef heifers. (3) Investigate mammary tumorigenesis in the estrogen inducible mammary tumor ACI rat model after long-term consumption of low level of bio-active Z containing experimental diets formulated from meat collected from Z- implanted beef heifers. The team of researchers assembles expertise in 3 areas to systematically study the mitogenic effects of Z in reproductive and hormone sensitive breast. These include modeling of in vitro and in vivo effects of Z (Lin), mapping and quantification of global and unique epigenetic alterations in gene promoters (Huang &Yan), and comparative quantification of global gene expression and identification of biomarkers (Wick). Significance: The proposed experiments will provide a clear linkage between bio-active dietary Z and PTPG down-regulation to eventual tumorigenesis via epigenetic regulation of the expression of PTPG and other tumor relevant genes. To translate our findings from bench top to bedside, selected biomarkers uncovered from the in vitro experiments will be further validated by proteomic analysis. It is our intent to provide comprehensive data (exposure levels during Z-implantation and after Z withdrawal from the beef heifers) to raise awareness of environmental exposure to the estrogen mimic, beef growth promoter, Z.